Developmental Cell Article Death Receptors DR6 and TROY Regulate Brain Vascular Development Stephen J. Tam,1 David L. Richmond,1 Joshua S. Kaminker,2 Zora Modrusan,3 Baby Martin-McNulty,4 Tim C. Cao,4 Robby M. Weimer,4 Richard A.D. Carano,4 Nick van Bruggen,4 and Ryan J. Watts1,* 1Neurodegeneration Labs, Department of Neuroscience 2Bioinformatics 3Department of Molecular Biology 4Biomedical Imaging Genentech, 1 DNA Way, South San Francisco, CA 94080, USA *Correspondence: [email protected] DOI 10.1016/j.devcel.2011.11.018 SUMMARY (Tam and Watts, 2010). For example, how does Wnt signaling regulate CNS angiogenesis and barriergenesis? Which mole- Signaling events that regulate central nervous cules downstream of Wnt signaling drive BBB development? In system (CNS) angiogenesis and blood-brain barrier addition to the canonical Wnt signaling components, these (BBB) formation are only beginning to be elucidated. studies also identified tight junction proteins and transporters By evaluating the gene expression profile of mouse that may contribute to mature BBB function (Daneman et al., vasculature, we identified DR6/TNFRSF21 and 2009, 2010). TROY/TNFRSF19 as regulators of CNS-specific We reasoned that gene expression profiling the BBB vascula- ture at embryonic, neonatal, and adult stages would enable angiogenesis in both zebrafish and mice. Further- identification of signaling molecules driving CNS vascular devel- more, these two death receptors interact both genet- opment. Subsquently, we show that death receptors DR6 and ically and physically and are required for vascular TROY are enriched in CNS vasculature during embryogenesis, endothelial growth factor (VEGF)-mediated JNK acti- and accordingly drive angiogenesis and barriergenesis in zebra- vation and subsequent human brain endothelial fish and mouse model systems. Using an in vitro cell sprouting sprouting in vitro. Increasing beta-catenin levels in assay, we demonstrate that these two tumor necrosis factor brain endothelium upregulate DR6 and TROY, (TNF) receptor family members are required for endothelial indicating that these death receptors are down- sprouting events in a cell-autonomous fashion through VEGF- stream target genes of Wnt/beta-catenin signaling, mediated JNK activation of angiogenesis. Additionally, we find which has been shown to be required for BBB that DR6 and TROY genetically and physically interact, and development. These findings define a role for death may form a coreceptor complex at the BBB. Finally, we identify canonical Wnt/beta-catenin signaling as a key transcriptional receptors DR6 and TROY in CNS-specific vascular regulator of DR6 and TROY BBB expression, suggesting one development. possible mechanism by which Wnt/beta-catenin transcription factor activity drives BBB angiogenesis and barriergenesis. INTRODUCTION RESULTS The vasculature of the CNS is an example of uniquely differenti- Identification of Genes Enriched in Brain Vasculature ated organ-specific blood vessels. CNS blood vessels differen- To identify signaling molecules driving BBB development, matu- tiate to form the blood-brain barrier (BBB), which consists of ration, and maintenance, we evaluated the gene expression a complex network of intercellular tight and adherens junctions profile of mouse vasculature at three distinct developmental that form a molecular seal between adjacent endothelial cells time points (Figure 1A), corresponding to CNS angiogenesis to limit molecular exchange across CNS vessels. Accordingly, (E14.5), astrocytic endfeet contact with cortical endothelium the BBB has many transport mechanisms that enable specific (P7.5), and maintenance of a mature BBB (Adult). We utilized jettisoning of unwanted molecules out of the CNS while import- florescence-activated cell sorting (FACS) to isolate CD31-posi- ing molecules essential for brain function (Rubin and Staddon, tive, CD45-negative endothelial cells from mouse brain cortices, 1999; Tam and Watts, 2010). liver, and lung vasculature (Figure S1A available online), and The molecular cues that regulate BBB development have subsequently verified FACS-mediated CNS vascular purification remained elusive until recent findings described a role for the by qPCR analysis (Figure S1B). As expected, microarray analysis canonical Wnt/beta-catenin signaling cascade in CNS angio- identified several Wnt/beta-catenin target genes and other previ- genesis and barriergenesis (Daneman et al., 2009; Liebner ously characterized BBB-specific components to be enriched in et al., 2008; Stenman et al., 2008). These discoveries now BBB vasculature (Figure 1C; Figure S1)(Daneman et al., 2009; raise a number of important questions related to BBB biology Hallmann et al., 1995; Pardridge et al., 1990). Developmental Cell 22, 403–417, February 14, 2012 ª2012 Elsevier Inc. 403 Developmental Cell Death Receptors Regulate CNS Vascular Development A BRAIN VASCULAR DEVELOPMENT C BBB markers E14.5 P7.5 Adult Glut1 P-gp Meca32 Lef1 4 1 5 4 2 0 4 ** 2 ** ** -1 ** 0 ** 3 ** ** ** -2 0 -2 ** 2 ** -3 GLUT1 -4 1 -2 -4 ** EPAEPA EPAEPA EPAEPA EPAEPA Log2 Expression Ratio Brain Liver/lung Brain Liver/lung Brain Liver/lung Brain Liver/lung D Novel transcripts enriched at BBB DR6 TROY Spock2 Adcyap1r1 GFAP GFAP 6 8 6 2 ** 6 ** ** 4 ** ** ** 4 1 ** 4 ** ** ** 2 2 2 ** ** 0 0 0 0 P-gp EPAEPA EPAEPA EPAEPA EPAEPA Brain Liver/lung Brain Liver/lung Brain Liver/lung Brain Liver/lung Adora2b Dlx2 Tspn5 Gpr37L 1 4 4 5 B Z-Score 0 2 E (E14.5) 3 4 Log2 Expression Ratio 0 ** P (P7.5) -1 ** ** -2 ** 2 ** 3 A (Adult) -2 ** ** -2 0 2 4 ** -4 ** 1 2 -6 Brain Liver/lung -3 0 ** 1 E14.5 P7.5 Adult E14.5 P7.5 Adult EPAEPA EPAEPA EPAEPA EPAEPA Brain Liver/lung Brain Liver/lung Brain Liver/lung Brain Liver/lung EPA E TNF receptor family members not enriched at BBB EP Tnfrsf10b Tnfrsf11a Tnfrsf1a E (E14.5) 0.5 2 1.5 P (P7.5) PA 0.0 1.0 0 ** A (Adult) -0.5 0.5 EA -1.0 -2 ** ** 0.0 ** ** -1.5 -4 -0.5 -2.0 E EPAEPA EPAEPA EPAEPA Log2 Expression Ratio Brain Liver/lung Brain Liver/lung Brain Liver/lung P A Developmentally Regulated BBB-enriched Genes **p < 0.005, * p < 0.05 Figure 1. Blood-Brain Barrier Expression Profiling Identifies Genes Enriched in Brain Vasculature (A) Three blood-brain barrier (BBB) developmental time points identified by immunohistochemical analysis of GLUT1 expression and astrocyte localization in the mouse cortex. (GLUT1, endothelial cells; GFAP, astrocytes; P-gp, P-glycoprotein) Results representative of at least three independent experiments are shown. (B) Distinct developmentally regulated classes of BBB-specific transcripts were identified by microarray analysis. Samples are in columns; probes are in rows. Red and green color indicates over- or underexpression of probes in brain tissue relative to liver/lung tissue of the same stage, respectively. Developmental stage is indicated to the left of the heatmap (E, embryo; P, pup; A, adult). The probes shown in the heatmap are also presented in Table S1. (C) Expression profiling FACS-purified BBB and liver/lung vasculature shows enrichment of BBB markers Lef1, P-gp, and Glut1. (D) Selected transcripts enriched at the BBB. (E) TNF receptor family members Tnfrsf1a, 10b, and 11a are not preferentially expressed in CNS vasculature. Box and whisker plots indicate distribution of data and highlight median (horizontal line through box) and the 25th and 75th quartiles (box edges). Expression values were compared between brain and liver/lung tissue at each developmental stage. Statistical significance was defined as those stage-specific comparisons yielding an adjusted p value (**p < 0.005; *p < 0.05). Further details of statistical analysis are described in extended Experimental Procedures. See also Figure S1 and Table S1. Microarray analysis across the BBB developmental timeline Further investigation of our gene expression data set yielded was able to resolve gene expression changes in a spatiotemporal several classes of BBB-specific transcripts: upregulated exclu- fashion (Figure 1B; Table S1; full microarray data set is publicly sively at E14.5, P7.5, adulthood, p7.5 and adulthood (e.g., available at the NCBI Gene Expression Omnibus). For instance, Tspn5 and Gpr37L), E14.5 and P7.5 (e.g., Adora2b and Dlx2), while the CNS-specific glucose transporter Glut1 is expressed in and across all three developmental time points (e.g., DR6, CNS at all three developmental time points, P-glycoprotein TROY, Spock2, and Adcyap1r1)(Figures 1B and 1D; Table S1). (P-gp) expression is highly upregulated at the BBB by postnatal Several of these transcripts have been previously described. day 7.5 (Figures 1A and 1C). Conversely, as Meca32 is only ex- For instance, both the proteoglycan SPOCK2 and homeobox pressed in fenestrated endothelium, transcript levels remained transcription factor DLX2 have been shown to be expressed in high in liver/lung vasculature at all time points and in embryonic CNS vessels and potentially drive CNS angiogenesis in a cell- brain vasculature, but are dramatically downregulated in brain autonomous fashion (Schnepp et al., 2005; Vasudevan et al., vasculature by P7.5 and adult time points (Figure 1C) (Hallmann 2008). Notably, the death receptors Tnfrsf21/DR6 and Tnfrsf19/ et al., 1995). TROY (Figure 1D; Figures S1D and S1E) were found to be highly 404 Developmental Cell 22, 403–417, February 14, 2012 ª2012 Elsevier Inc. Developmental Cell Death Receptors Regulate CNS Vascular Development expressed in CNS-specific vasculature, while several TNF confocal live imaging of the CNS. Whereas control, Adcyap1r1, receptor family members including Tnfrsf10b, Tnfrsf11a, and Tspn5, and Tnfrsf1a morphant embryos excluded most of these Tnfrsf1a are not enriched in BBB vasculature (Figure 1E). tracers, as rhodamine-dextran was restricted to CtA vessel lumen while DAPI-stained nuclei were only found in CtA vessels, Zebrafish Candidate-Based Genetic Screen Identifies DR6, TROY, and Spock2 morphants exhibited dramatic leakage Regulators of CNS Angiogenesis and Barriergenesis of both tracers across the BBB, as evidenced by an increase in To understand the functional relevance of the BBB-specific tran- rhodamine-dextran signal and parenchymal nuclear staining by scripts identified in our expression profiling analysis, we em- DAPI (Figure 2A).